Process for the production of antibiotic substance cephemimycin

ABSTRACT

Process for the production of a known antibiotic substance, cephemimycin, by cultivation of a newly discovered microorganism Streptomyces jumonjinensis strain No. 3008 according to any method employed for Streptomyces. The antibiotic substance is effective against gram-negative bacteria.

United States Patent [191 Arai et al.

[451 Feb. 11,1975

[ PROCESS FOR THE PRODUCTION OF ANTIBIOTICSUBSTANCE CEPHEMIMYCIN [75] Inventors: Mamoru Arai; Yashuhiro ltoh;

Masaki Nakahara; Hisashi Kayamori; Shinichi Sugawara, all of Tokyo, Japan [73] Assignee: Sankyo Company Limited, Tokyo,

Japan 22 Filed: Aug. 16, 1973 211 App]. No.: 389,009

[30] Foreign Application Priority Data Aug. 31, 1972 Japan 47-87339 [52] U.S. Cl 195/80 R [51] Int. Cl Cl2d 9/00 [58] Field of Search l95/80 R [56] References Cited UNITED STATES PATENTS 3,80l,464 4/1974 Gorman et al. l95/8O R Primary ExaminerLionel M. Shapiro Assistant Examiner-Robert J. Warden Attorney, Agent. or FirmFlynn & Frishauf [57] ABSTRACT Process for the production of a known antibiotic substance, cephemimycin, by cultivation of a newly discovered microorganism Streptomyces jumonjinensis strain No. 3008 according to any method employed for Streptomyces. The antibiotic substance is effective against gram-negative bacteria.

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6 LO 3 N NU MBER OF WAVES PROCESS FOR THE PRODUCTION OF ANTIBIOTIC SUBSTANCE CEPHEMIMYCIN This invention relates to a new process for the production of an antibiotic substance, cephemimycin.

More particularly, this invention is concerned with a new process for producing the above antibiotic substance by cultivation of a newly discovered microorganism.

We have found that the antibiotic substance, cephemimycin, is produced in a cultured broth of Streptomycesjumonjinensis strain No. 3008 which was isolated from a soil sample collected at Jumonjitoge, Chichibu, Saitama Prefecture, Japan. This antibiotic is isolated from the cultured broth, purified and characterized that it is highly effective against gram-negative bacteria, particularly streptomycin-resistant, kanamycinresistant and multi-resistnat Escherichia, resistant Klebsiella and multi-resistant Proteus.

The antibiotic substance, cephemimycin, which is produced by the process of this invention has been found to have the following chemical structure and multi- 20 OCH 2 COOH l CODE 0 Morphological characteristics of the above cephemimycin-producing strain No. 3008 are as follows: 1. When observed under a microscope, aerial mycelium is well-branched and forms tightly closed spirals, but no conidia chains are observed. Spores are cylindrical in shape and 0.6 0.8 X 1.0 1.2 p. in size. Spore surface is smooth. Spore chains are in 10 50 spores per spore chain. No flagellate spore and sporandium are formed and sporophores are on aerial mycelium.

2. Results obtained in the culture on various culture media (observation made after a 2-week cultivation at identified to be the same substance as that named An- 25 28C unless otherwise stated) are as shown in Table 1.

Table 1 Aerial Substrate Soluble Medium Growth mycelium mycelium Reverse pigment Sucrose- Moderate Poor, Poor, While None nitrate white colorless agar Glucose- Good Moderate Poor, Light asparagine brownish yellowish brownish do. agar rey grey grey Glycerol- Moderate Poor, Abundant, Pale asparagine white yellowish yellowish do. agar grey brown Inorganic Good Poor, Moderate Pale saltsgreyish yellowish yellow do. starch white grey agar Tyrosine Moderate None Moderate Pale agar colorless yellow do. Nutrient Good Poor, Moderate Pale agar white yellowish yellow do. medium grey Yeast Abundant Abundant, Abundant, Dull Pale extractwhite to yellowish yellow yellowish malt extract brownish grey brown agar grey Oatmeal Moderate None Moderate Colorless None agar colorless tibiotic Substance A-l6886l in Dutch Pat. No. 3.Phys1olog1calproperties of the strain No. 3008 are 7011805 and also that named Antibiotic Substance 55 shown in Table 2.

Milk peptonization:

Melanin formation:

Nitrate reduction:

37C., no growth 25C., 7 days (pH 6.4)

37C., no growth (in tyrosine-agar medium) (in peptone-yeast extract iron agar medium) 4. Carbon source utilization pattern of the strain No. 3008 on Pridham-Gottliebs agar medium is shown in Table 3.

From the above, the strain No. 3008 has been identified as a new species distinctly different from Actinomyces mutabilis and named Streptomyces jumonjinensis Table 3 5 strain No. 3008. The strain No. 3008 has been deposited under an accession No. 1545 with Technical Reo ac oge search Institute of Microbial Industry, Agency of Indusjg gzi 1 $312, 22? trial Science & Technology, Ministry of International D-galactose Rafi 'mose Trade and Industry, Japan, and also as NRRL-574l in 323222? Egg? 1 the Northern Regional Research Laboratory, Northern L-rhamnose D-mannitol Central Region, Agricultural Research Service, United Sucrose States Department of Agriculture, at Peoria, 111., U.S.A. Although we have explained the strain No. 3008 in From the Summary of the above Prepemes, the Stfam the foregoing, it is well-known that various properties 3008 belongs to genus Steptomyees, its myeellum 5 of Streptomyces are not definite, but may easily be varf rm g y close p spores are cylmdrlcal m ied naturally and artificially. The strains which may be shape and p f Surface P Celorless t0 yellowish employed in this invention include all of the strains y growth 15 Observed Varlous media, White 10 which belong to the genus Streptomyces and are capabrownish grey aerial hyphae are formed, soluble pigbl f producing h i i ments are scarecely formed to the extent that some F comparison on various properties of the strain pale yellowish brown soluble pigments are only ob- No. 3008 with the microorganisms known'to produce served in a yeast extractmalt extractagar medium. the same antibiotic substance of cephemimycin, more Also, pale yellow to light brownish grey colors are obspecifically, Streptomyces clavuligerus in Dutch Pat. served on the reverse of the collony. As to physiolog- No. 7011805 and International Journal of Systematic ical properties, no melanin formation, starch hydrolysis Bacteriology, 21, 4, 326, 1971 as well as Streptomyces and milk coagulation are observed. lactamdurans in the publicly opened specification No. Searching the known strains having the above prop- 3286/1971 of Japanese Patent Application, are found erties, as the most closely related strain may be menthe following points. tioned Actinomyces mutabilis which was described in l. Morphological characterlstlcs (After 14 day culthe International Journal of Systematic Bacteriology, u Vol. 18, No.2,148 150 (1968). However, when compared with the disclosure in the above literature and Strain the strain ISP No. 5169 which IS the authentlc strain of Character- Sfrepfgmyces Sirepmmyce; Streptomyces Acrinomyces mutabilis, the strain No. 3008 of this inislics jumonjinemis vlflvuligems lavlamdumm vention differs from them in the following points shown in Table 4. Branching Monopodial Sympodial Straight with T bl 4 of aerial branching branching few branching a e mycehum (Club-shaped S h S sidebranches Nature Strain No. 3008 Strain ISP No. 5169 Spore l0 50 per 1 4 per None Shape, Cylindrical, smooth, Cyllndrlcal, smooth, hai hain hai surface and 10 5O spores 3 10 spores Fra llar N No N number of spore spores Sporangium None None None Spirals Compact Spiral filaments Spore Smooth Smooth None spiral filaments or hook filaments surface (Cylindrical) (Oblong to Color of White to brownish White to grey cylindrical) aerial grey Site of Only on Only on aerial mycalium sporophore aerial mycelium Coremia fonnation mycelium formation Sporephore Spiral Spiral or hook Carbon D-xylose, D-fructose, D-xylose, 5O utilization D-mannitol not D-fructose,

utilized D-mannitol utilized Antibiotic Cephemimycin 2. Cultural characteristics on various agar medla. Pmducm" (After 14 day culture) Streptomyces Streptomyces Streptomyces jumonjl'nensis clavuligerus lactamdurans Sucrose- G: Moderate Scant Moderate nitrate AM: Poor, White Creamy white agar R: White Deep cream SP: None None Glucose- G: Good Moderate asparagine AM: Good; Olive Good; White y agar R: Yellowish Pale gray yellowish green SP: None None Glycerol- G: Moderate Good Moderate asparagine AM: Poor; White Good; White Cream agar R: Pale yellow Pale Golden to yellowish orange green SP: None None Pale amber C ontmued Strepromyces Sireplomyces Sm' mmwux jummriinenris clm'uligerus Iacrinudu riins Inorganic G: Good Abundant Moderate salts- AM: Poor: Light Good: Medium White to green gray gray to cinnamon starch R: Pale Grayish Cream to yellowish brown yellow greenish orange agar SP: None None None Nutrient G: Good; Good; Moderate agar AM: Poor; White Sparse; White Cream R: Pale Pale Golden yellowish yellowish brown green SP: None None None Tyrosine G: Moderate Moderate Moderate agar AM: None Poor; Cream and Yellowish white y R: Pale yellowish Pale yellow Tannin to brown orange SP: None None None Yeast G: Abundant Abundant extract- AM: Abundant; Abundant; malt White Light grayish extractolive agar R: Dull yellow Grayish yellow SP: None None Oatmeal G: Moderate Moderate Moderate agar AM: None Good; White Cream R: Colorless Pale yellow Orange SP: None None None G: Growth AM: Aerial Mycelium R: Reverse SP: Soluble pigment Tomato-pastc-oatmeal agar medium for Slrepm/nycex laclumduruns 3. Physiological characteristics.

Strepwmyces Streptomyces Streptomyces jumonjinensis clavuligerus lactamdurans Tyrosinase reaction Nitrate reduction Starch hydrolysis Gelatin liquefaction Melanin formation Peptone-yeast extract iron agar Tryptone-yeast broth lilll Not described l++ll Not described significance in classification of actinomycetes, and also of a great difference in carbon utilization between Cultivation in the process of this invention may be carried out according to the method generally employed for Streptomyces. Shaken culture or submerged culture in a liquid medium is preferable.

As medium components may be employed any of the well-known nutrient materials for Srreptomyces. For instance, as an assimilable carbon source, glucose, glycerol, maltose, dextrin, starch, soybean oil, cotton seed oil, etc. and, as an assimilable nitrogen source, soybean From the above comparative studies on physiological and morphological properties, it can be clearly concluded that Streplomyces jumonjinensis in this invention is distinctly different from Streptomyces lactamdumm from the physiological and morphological points of view, and also that the present microorganism is distinctly different from Streptomyces clavuligerus in view of different branching of aerial mycelium, which is of meal, peanut meal, cotton seed meal, fish meal, corn steep liquor, peptone, meat extract, yeast, yeast extract, sodium nitrate, ammonium nitrate, ammonium sulfate, etc. may be used. And, sodium chloride, phos phates, calcium carbonate, etc. may be used as an inorganic salt. A minor amount of a metal salt may also be added, if necessary.

In carrying out liquid cultivation with aeration and agitation, an anti-foaming agent, e.g., silicon oil, vegetable oils, surfactants, etc. may be suitably employed.

The pH of the medium may be suitably within or around neutral range and cultivation temperature may be usually of 25 30C., in particular about 27C. being preferred.

Change with time lapse in the activity of an antibiotic substance cephemimycin, which is being produced in the cultured broth as the cultivation proceeds, can be determined by a well-known cylinder-plate test method using Proteus vulgaris as a test microorganism. Usually, the maximum production of cephemimycin may be accomplished by cultivation for 40 80 hours. For instance 40 pg/ml. of cephemimycin in a 30 ml. volume jar fermentor was produced in the cultured broth after 40 hours.

Cephemimycin is easily soluble in water and existed predominantly in a liquid portion of the cultured broth.

After completion of the cultivation, mycelium and other solid mass are removed by a filtration procedure using diatomaceous earth and the like as a filter aid or by centrifugal separation and cephemimycin involved in the filtrate or supernatant phase may be isolated and purified by utilizing its physico-chemical properties.

For instance, cephemimycin may be recovered from the cultured broth by the use of an adsorbent. As the adsorbent may be, for example, active charcoal powder and the cephemimycin adsorbed by active charcoal powder may be eluted with methanol-water, n-butanolwater, aqueous acetone and the like. Also, cephemimycin is an amphoteric substance having both strong acidity and weak basicity and thus may be adsorbed and eluted with cation or anion exchange resins.

Examples of the cation exchange resin which may be employed are strongly acidic cation exchange resins, e.g., Dowex 50W X 4 (manufactured by Dow Chemical Co., U.S.A.), Amberlite lR l2O (manufactured by Rohm & Haas Co., U.S.A.) and the like. As examples of the anion exchange resins may be strongly basic anion exchange resins, e.g., Dowex lxl (manufactured by Dow Chemical Co.), etc., but weakly basic anion exchange resins, e.g., Duolite A-2 (manufactured by Diamond-Alkali Co., U.S.A.) maybe preferably employed. Further,,a chromatography may be effected by the use of silica gel, Avicel (microcrystalline form cellulose, available from Asahi Kasei Kogyo 1(.K., Japan), etc. Cephemimycin may be purified too a single isolated state on a thin layer chromatography by the repeated use of any combination of such purification means. Physico-chemical properties of cephemimycin are given below.

1. Appearance Pale yellowish white powder 2. Melting point 160C. (with decomposition) 3. Elementary analysis C 42.60% H 5.76% N: 11.42% S 5.52%

4. Specific rotation [01],, 102 (c 1, water) 5. Neutrality, basicity or acidity pKa 2.97, 9.41

6. Ultraviolet absorption spectrum As shown in FIG. 1 values of maximum absorption positions and of E, 0,, are as shown in Table 5.

Table 5 Absorption Max. Absorption Max. Solvent u) 1 rm (m 1 rm H 0 238 114 263 130 0.1N HCl 244 98 263 108 0.lN NaOH 229-230 162 7. Infrared absorption spectruum As shown in FIG. 2 (measured with KBr tablets) 8. Solubility It is easily soluble in water, soluble in methanol, sparingly soluble in ethanol and insoluble in other organic solvents.

9. Color reaction Positive for ninhydrin, Benedict and Tollens reactions.

Negative for ferric chloride, biuret, orcinolhydrochloric acid, Anthrone and Elson-Morgan reactions.

10. Stability lts aqueous solution is relatively stable at an acidic or slightly alkaline state and and 85 of its activities remain unchanged at pH 2, 7 and 8, respectively, even after heat treatment at 60C. for 30 minutes.

11. Thin layer chromatography By a thin layer chromatography (ascending method) using Cellulose Chromatogram Sheet 6065 (manufactured by Eastman Kodak Co., U.S.A.), Rf values are as shown in Table 6. Bioautography with Pro teus vulgaris or coloration with ninhydrin was employed for detection.

Table 6 Developing solvent Rf value n-butanol: acetic acid water 40% n-propanol 0.83 3 amrsnonlipm chloride methanol 0.50 n-propanol pyridine acetic acid water 0.29

methyl ethyl ketone n-butanol water 12. High voltage paper electrophoresis Electrophoresis is effected for 30 minutes under a condition of 3,300 V and 40 mA with a buffer solution of formic acid acetic acid water (1 3 36) having pH 1.8 by the use of Toyo Filter Paper No. 5 with 60 cm X 20 cm (manufactured by Toyo Roshi K.K., Japan) and detection is made in the same manner as in the thin layer chromatography. The mobility of cephemimycin is 0.3, when that of L-alanine is 1.0.

The powdery cephemimycin having the above physico-chemical properties is further purified by a column chromatography to give white powder, which has the same properties as given in the foregoing except for the physical properties shown below.

1 Elementary analysis (as monoammonium salt) C 41.69%, H 5.86%, N: 14.37%, S 6.88%

Calculated: C 41.46%, H 5.44%, N: 15.11%, S:

6.92% 2. Specific rotation [a] (c 1, H O) 3. Ultraviolet absorption spectrum Solvent Absorption Max. E

10 group consisting of 10 animals, were inoculated with l X 10 cells per mouse of Escherichia coli 640, a multi-resistant strain, and cephemimycin was subcutaneously administered in its sterile so- 5 dium chloride solution at 6.25 50 mg./kg. two times, namely, immediately after the inoculation H20 242 132 and after 4 hours from the inoculation. The num- 52g bers of surviving mice are as shown in Table 8. 0.1N HC] 263 133 Table 8 Day after administration l 2 3 5 7 Dose (mg/kg.) Biolo ical activities of ce hemim cin are set forth' b l g p y 0 (Control) 10 4 0 0 o 0 Bow 6.25 10 7 6 s 6 10 l. Antimicrobial spectrum 12.5 10 10 9 9 9 9 Minimal inhibitory concentrations of various micro- 10 10 m 10 10 10 10 1t) 10 10 10 organisms are as shown in Table 7.

Table 7 Test organism Medium* MIC ug/ml.

Staphylococcus aureus 209? JC-2 H 200 S. aureus 56 do. 200 S. aureus 193 do. 200 S. aureus 52-34 do. 200 Bacillus subrilis PCI 219 do. 12.5 Sarcina [urea PC1 1001 do. 25 Corynebacrerium xerosis B58-3 do. 25 Mycobacrerium smegmatis ATCC 607 G 200 Escherichia cali NIHJ JC-2 do. 12.5 E. coli (SM resistant) G 3.12 E. cali (KM resistant) do. 0.78 E. coli GN2645 (Multi-resistant) do. 12.5 Klebsiella pneumoniae PCI 602 do. 3.12 K. penumoniae GN2984 (Multi-resistant) do. 6.25 Proteus vulgaris OX 19 do. 3.12 P. reugeri GN2825 (Multi-resistant) do. 50 P. mirabilis IFO 3849 do. 3.12 P. mirabilis ON 829 (Multi-resistant) do. 3.12 P. mirabilis GN1973 do. 12.5 P. mirabilis GN 1973 RG-2984 (Multi-Resistant) do. 3.12 Pseudomonas aeruginasa B l-l do. 12.5 P. aeruginora SC-8753 do. 200 P. Sp. SC-8328 do. 200 Candida albicans YU 1200 S 200 C. lropicalis WII 42 do. 200 Saccharomyces cerevisiae ATCC 9763 do 200 Cryptococcus neoformans WH 15-4 do. 200 Trichophymn menlagrophyres F 63-9 do. 200 Epidermophyton fluoccasum ATCC 10227 do. 200 Penicillium chrysagenum Q 176 P 200 Aspergillus oryzae lAM 2630 do. 200 Furarium moniliforme IAM 5062 do. 200 Batrytis cinerea IAM 5126 do. 200 Piricularia oryzae IAM 5087 do. 200 Pellicularia sasakii P-35 do. 200

Medium H: Heart infusion agar 0: Heart infusion agar with 1% glycerol 5: Sabouraud-dextrose agar P: Potato-dextrose agar Cultivation: MIC was determined by that obtained after As is apparent from the Table 8, cephemimycin can the cultivations for 24 hours at 37C. for bacteria and exhibit its protective effect even at a dose of 6.25 for 48 hours at 28C. for yeasts and fungi. As a result mg./kg. of the MIC on bacteria in Heart Infusion medium with From the results of our studies on known antibiotic l0 horse serum, no effect has been found upon the substances having the above-described physicoaddition of horse serum. chemical and biological properties, it has been con- As is apparent from the Table 7, cephemimycin has firmed that the cephemimycin obtainable by the presa high antibacterial activity against gram-negative bacent process is identical with the above-mentioned antiteria. biotic substances A-16886I and 842A.

2. Toxicity The process of this invention is more illustratively ex- No abnormal phenomena have been observed after plained by the following examples, but the production 14 days from administration of 800 mg./kg. to according to this invention may be: practised in various mouse by intravenous injection. manners based upon the properties of cephemimycin as 3. Protective activity Cephemimycin has been observed to show an effectiveness in a protection test on mouse by Escherichia coli. More specifically, ddY-strain mice of each average body weight of about 20 g., each disclosed herein. Then, this invention is not limited to these examples but includes all of the methods for production, extraction and purification of cephemimycin by well-known means upon the thus proved properties of cephemimycin through this invention.

EXAMPLE 1.

The strain No. 3008, which had been cultivated on a malt extract-yeast extract agar medium, was inoculated to 100 ml. (in a 500 ml.-volume Sakaguchi flask) of a medium (of pH 7.2 before sterilization) containing 2.0 glucose, 1.0 starch, 0.9 yeast, 0.5 Polypepton, 0.5 meat extract, 0.3 calcium carbonate, 0.5 sodium chloride and 0.02 Disfoam CB442 (available from Nippon Oils & Fats Co., Ltd., Japan) as an antifoamer and a shaking culture was effected at 27C. for 2 days to make a seed culture. 200 ml. of this seed culture was placed into a 30 1.-volume jar-fermentor containing l. of the culture medium of the same composition as set forth above and cultivation was effected for 40 hours at a rotation of 200 rpm, an aeration of 15 l./min., an inner pressure of 1.1 kg/cm and a temperature of 27 i 1 C. to produce 40 pg/ml. of cephemimy- 14 l. of the cultured broth was filtered by the use of diatomaceous earth (Celite 545; trade name) as a filter aid and combined with aqueous washings to form 12 l. of the filtrate. 300 g. of active charcoal powder was added to the so obtained culture filtrate and stirring was done, whereupon cephemimycin was adsorbed thereon. Filtration was again effected by the use of diatomaceous earth and washing was made with water. For the elution of cephemimycin from the active charcoal powder, 80 acetone was used in two portions of 7.5 1., the active charcoal powder was removed by filtration and then the eluate was concentrated under reduced pressure to give 250 ml. of a concentrate (41.3 of a yield on activity). The concentrate was passed through a column packed with 400 ml. of Duolite A2 (C 1 -type) to adsorb cephemimycin therein, the column was washed with 650 ml. of water and eluted with 0.1 N hydrochloric acid. 650 ml. of the active eluate (yield 34.3 was neutralized with 0.1 N aqueous ammonia, 13 g. of active charcoal powder was added thereto for de-salting, thereby making cephemimycin adsorbed, and then elution was made twice with 350 ml. of 80 aqueous acetone (yield 1 7.2 The eluate was concentrated to ml. under reduced pressure and freezedried to give 950 mg. of crude powder (activity of 86.2 mcgu./m g. The crude powder was dissolved in a small amount of 80 aqueous methanol and adsorbed on a column prepared with 300 g. of silica gel, Mallinckrodt CC-4 (manufactured by Mallinckrodt Chemical Works) in chloroform: methanol: water (9 9 2). Elution was effected with the solvent system of the same composition as shown above. 180 ml. of active eluate was collected, concentrated to dryness under reduced pressure, dissolved in 50 ml. of water, extracted twice with ethyl acetate in equal portions to remove contaminates and then freeze-dried to give 81 mg. of cephemimycin (activity of 250 mcgu./mg.) as crude powders (Yield 4.2

EXAMPLE 2 2 l. of cephemimycin-containing eluate obtained from Duolite A2 by the same procedure as given in Example 1 (a yield on activity of 31.2 was neutralized with 1 N aqueous ammonia, adsorbed on a column of 46 g. of active charcoal for chromatography (manufactured by Wako J unyaku K.K., Japan) packed with water. washed with 500 ml. of water and then eluted with 8 aqueous acetone. 1 l. of the active eluate (yield 22.6 was passed through a column of 300 ml. of Dowex 50W X 4 (equilibrated with 0.01 M sodium citrate of pH 3) to adsorb cephemimycin therein. which was washed with 600 ml. of the same buffer solution and then eluted with 0.03 M sodium citrate of pH 3. 500 ml of the active eluate (yield 18.7 was collected and desalted by conducting again a chromatography with active charcoal under the same condition as mentioned above. ml. of the eluate from 8 %aqueous acetone was concentrated to 20 ml. under reduced pressure and freeze-dried to give 79 mg. of cephemimycin as pale yellowish white crude powders (activity of 527 mcgu./mg.) (yield 9.4

EXAMPLE 3 In a 600 l-volume fermentation tank was prepared 300 l. of a liquid culture medium having the same composition as shown in Example 1. After sterilization, 50 l. of a seed culture, which had previously been cultivated in a medium having the same composition for 24 hours, was inoculated and cultivation was effected at 27C for 45.5 hours (aeration of 300 l./min., rotation of 190 rpm, inner pressure of 1 kg./ cm). 350 l. of the culture broth obtained by the above cultivation was filtered through a filter press by the use of diatomaceous earth as a filter aid to give 310 l. of culture filtrate. To the so obtained culture filtrate were added 6.2 kg. of active carbon powder and 6 kg. of diatomaceous earth and, after stirring, filtration was made to collect active charcoal powder. The charcoal cake was washed with water and extracted twice with l. of 80 aqueous acetone. l. of the extract was concentrated under reduced pressure to give 42 l. of a concentrate (yield on activity of 47.0 The concentrate was adjusted to pH 8 and passed through a column of 25 l. of Duolite A-2 (acetateform). By this procedure, 80 of active component was passed therethrough (yield 37.6 The effluent was combined with the aqueous washing to amountto 92 l. to which 1.84 kg. of active charcoal powder was added. After stirring, the cephemimycin adsorbed was extracted twice with 45 l. of 8 aqueous acetone to give 70 l. of extract, which was then concentrated to 3 1. under reduced pressure (yield 23.0 The concentrate was diluted to 6.1 with water, adjusted to pH 4.15 with acetic acid and passed through a column of 3 l. of Duolite A2 (acetate-form).

After washing with 15 l. of acetic acid of pH 3.0, cephemimycin was eluted with a 0.2 N pyridineacetic acid buffer solution of pH 5.5. The active eluate (l 8 l.) concentrated to 900 ml. under reduced pressure (pH 3.0, yield 20.7 From the concentrate, cephemimycin was again adsorbed on a column of 1.65 l. of Duolite A-2 (acetate-form), the column of which was then washed with 7.5 l. of acetic acid of pH 3.0 and eluted with the above-mentioned pyridine acetic acid buffer solution. 3 l. of the active eluate was concentrated to dryness under reduced pressure (yield 20.0 The crude powder was washed twice with 300 m1. of ethanol and dissolved in 300 ml. of water (yield 18.4 660' mg. of active charcoal powser was added thereto, followed by stirring and filtering (yield 17.3 'The filtrate was concentrated to 30 ml. The concentrate was passed through a column of 900 ml. of Amberlite CG-50 (H-form) to remove a trace of remaining pyridine through adsorption, the column of which was washed with water 1g the effluent was concentrated followed by freeze-drying to give 3.94 g. of cephemimycin in a pure state (1,000 mcqu./mg.) with pale yellowish White color (yield 12.7

What is claimed is:

l. A process for producing an antibiotic substance cephemimycin sis strain No. 3008 (NRRL 5741) in a nutrient medium and recovering the antibiotic substance from the cul tured broth.

2. The process according to claim 1 wherein said cultivation is effected under aerobic condition.

3. The process according to claim 1 wherein said cultivation is effected at a temperature ranging within 25C. to 30C.

4. The process according to claim 1 wherein said cultivation is effected for 40 hours.

5. The process according to claim 1 wherein said medium has a pH value of about 7.

I PAGE ONE UNITED STATES PATENTANI) TRADEMARK OFFICE vCERTIFICATE OF CORRECTION Q PATENT NO. 1 3,865,693

DATED February 11, 1975 |NV ENTOR(S) MAMoRU AR I r; al

It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 1, line 20: after "multi", replace "resistnat" with resistant Columns l-2, Table under "Reverse", "While" should be White Columns 5-6, "Physiological characteristics" Table:

next to "Milk peptonization" the last two columns should read as follows:

-- (temperature (alkaline) not described) (temperature (temperature not described) not described) Column 7 last line: replace "E with Ei% cm.

Column 8 Table 5, and Column 9, first table: in headings, a 1 a n n replace E Cm. with E cm.

Column 8 line 10: replace "spectruum" with spectrum Q Column 9, line 9: "0.1 N HCl" should be printed opposite "245" and "117". (one line above its present position) Column 9, Table 7: replace "fluoccosum" with floccosum v PAGE TWO UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. I r 3,865,693

DATED February ll, 1975 INV ENTOR(S) I MAMORU ARAI et al it is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 10, Table 8, second line: under "(Control)", delete "10"; in the same line, the remaining numbers should read: l0 7 6 6 6 6 Column 12, line 48: replace "6.1" with 6 l.

Column 12, line 66: replace "powser" with powder Swgncd and Sealed this twenty-fifth a O N [55M] D y f ovemberl975 Arrest:

RUTH C. MAISON C. MARSHALL DANN :llfijflllg Officer (ummr'ssr'vner 11] Parents and 'l'rarlvmurks 

1. A PROCESS FOR PRODUCING AN ANTIBIOTIC SUBSTANCE CEPHEMIMYCIN
 2. The process according to claim 1 wherein said cultivation is effected under aerobic condition.
 3. The process according to claim 1 wherein said cultivation is effected at a temperature ranging within 25*C. to 30*C.
 4. The process according to claim 1 wherein said cultivation is effected for 40 - 80 hours.
 5. The process according to claim 1 wherein said medium has a pH value of about
 7. 